Based significantly on the efforts of both intramural and extramural research supported by the National Cancer Institute (NCI) and the National Institutes of Allergy and Infectious Disease (NIAID), adoptive cellular therapy (ACT) of cancer and chronic viral infections with tumor-infiltrating and receptor-engineered T cells has moved from experimental setting to one of increasing clinical reality. Although transfer of high-affinityT cells holds great promise for a variety of tumor types, their supraphysiologic affinities and antigen sensitivity can cause significant on-target immunpathologic damage to self-tissues that share expression of the targeted antigen. A key goal for expanding the efficacy and range of tumors treatable by ACT would be realized by devising ways of controlling the balance between anti-tumor and anti-self-immunity to favor the former. The goal of this exploratory project is to determine whether CD8+ T cells expressing moderate-affinity receptors can be induced to specifically eradicate tumors through immunologic manipulation of their tumor microenvironment without autoimmune damage to healthy self-tissues where such alterations are absent. This work will be carried out in a murine model of ovarian carcinoma (ID8) which features shared expression of a model antigen with both pancreas and kidney in RIP-mOVA mice. ACT will be performed in ID8 tumor-bearing RIP-mOVA mice using moderate affinity OT-3 CD8+ T cells specific for the shared antigen that mirror the post-tolerant repertoire in humans in that they have undergone negative selection and remain ignorant of their cognate peripheral antigen unless they are specifically primed to do so. Aim I will define the therapeutic parameters of tumor control versus on-target autoimmunity by moderate-affinity CD8+ T cells, with the working hypothesis that the threshold for reactivity to both tumor and self is closely coupled. These experiments will examine the role of cell number and priming stimulus in inducing tumor control versus autoimmune destruction of beta islets. Aim II will evaluate whether alterations of tumor microenvironment can allow a number OT-3 cells that is below the threshold for autoimmunity to selectively recognize treated and distal tumors, with the working hypothesis that local immunomodulation can open a window of opportunity for tumor control by moderate affinity T cells. Modifications to be tested in this regard include A) removing inhibitory cell types (regulatory T cells, type 2 macrophages, myeloid-derived suppressor cells) or signals (TGF-?, IL-6, IL-10, PD-1/L1, and CTLA-4), B) enhancing local OT-3 activation with intratumoral immunostimulatory cytokines (IL-2, IL-12) or costimulatory signals (CD27, 4-1BB, CD40), and C) increasing local homing and retention of OT-3 cells by intratumoral injection of CXCR3 chemokines (CXCL9/10). If successful, this project will support a new paradigm that will enable a greatly expanded repertoire of tumor-specific TCRs to be used in ACT, and will greatly decrease the occurrence of on-target toxicities that have limited its application to a broader range of tumor types.